| PrefaceMultiple epiphyseal dysplasia ( MED) is a rare inherited osteo-chondrodysplasia, resulted from defects of structural proteins in extracellular matrix of cartilage and transport protein in cytomembrane. It is a genetically heterogeneous disorder with marked clinical variability. The main inherited mode is autosomal dominant trait with high penetrance, rare autosomal recessive. Three clinical phenotypes, including milder (Ribbing type) ,severe (Fairbank type) and unclassified MED have been described. However, there is no definite criteria to differentiate them. MED is characterized, with onset in early childhood , by mild to moderate short stature and early - onset osteoarthri-tis. The symptoms consist of pain and stiffness of the large joints,waddling gait, short stubby hands, genu varum or valgum. Necrosis of femoral head can be found in 309c patients. In 1994 ,one of the disease genes was localized to 19p ' , subsequently found that mutations of the gene encoding cartilage oligorneric matrix protein ( COMP) were responsible for MED. So far , three genetic loci for MED had been confirmed, i. e. the genes encoding the a2, a3 chain of type DC collagen and encoding diastrophic dysplasia sulfate transporter (DTDST). 57 percent of the patients reported abroad resulted from mutations in COMP gene. Nine missence point mutations, one insertion and onedeletion in COMP gene exist for MED. These mutations distribute in six exons. Among them , the rate of mutation in exon 10 is high. So far no report about the molecular pathogenesis study inland.To search the mutation in exon 10,13 and 14 of COMP gene in Chinese patients with MED, ten patients ,ten parents and thirty normal control were examined by PCR - SSCP and direct sequencing.MaterialPremiers of PCR were synthesized by CELL Corporation ;Taq enzyme, dNTPand 10xBuffer were from Takara Corporation; DNA Gel Extraction Kit was from Sangon Corporation.Method1. Subject and specimens Our study was done with samples of blood that were collected in two groups. One group included eight sporadic patients with MED and a family and ten parents. The other group were thirty normal unrelated children. Venous blood were drawn from all the subjects, and genome DNA was extracted from white blood cells according to the standard procedures. 2. PCR amplification DNA was amplified in 25 - ul PCR reaction. Samples were amplified in a PTC200 thermal cycler with an initial denaturation at 94C for 8 min ;followed by 30 cycles of denaturation at 94C for 40 s,annealing that varied ,depending on the exon,from 55C to 57C for 40 s,and extension at 72C for 40 s, with a final 7 - min extension at 72C. PCR products were dected by 2% agrose gel. 3. SSCP analysis 5 - ul products was then diluted 1:1 with loading buffer. The mix was heated at 96C for 10 min, plunged into ice, and immediately loaded on nondenaturing 8% polyacrylamide gels (20 x 18 x0. 75cm,49 ac-rylamide: 1 bis) in 1 x Tris - Borate - EDTA Buffer. The gels were run at 10 - 14C for 4 -5 hours at 300V. All the SSCP gels were developed by silver staining. 4. Sequencing PCR products with mobility shift were cleaned by using of DNA Gel Extraction Kit . Direct sequencing was done on ABI PRISMTM 377XL DNA Sequencer in TaKaRa Corporation.Results1. PCR - SSCP analysis All the normal control had the same band . Among the patients and parents, only a sporadic case of severe type exhibited single strand mobility shift in the product of exon 14 . His mother with normal pheontype displayed the same band as normal control. 2. DNA sequence analysis DNA sequencing confirmed that an homozygous G( +22)-A transition existed in the 5' end of intron 14, whereas nucleiotide sequence of exon 14 is the same as that from Genbank.DiscussionCOMP is a Calcium - binding protein which is expressed prominently in the territorial matrix surrounding chondrocytes. It is a 524 -KD homopentameric glycoprotein , containing an amino - terminal domain , four contiguous epidermal growth factor - like repeats, eight contiguous c...
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